Rational approaches for the design of various GABA modulators and their clinical progression

Abstract

GABA (γ-amino butyric acid), is an important inhibitory neurotransmitter in the central nervous system. Attenuation of GABAergic neurotransmission plays an important role in the etiology of several neurological disorders including epilepsy, Alzheimer’s disease, Huntington’s chorea, migraine, Parkinson’s disease, neuropathic pain, and depression. Increase in the GABAergic activity may be achieved through direct agonism at the GABA_A receptors, inhibition of enzymatic breakdown of GABA, or by inhibition of the GABA transport proteins (GATs). These functionalities make GABA receptor modulators and GATs attractive drug targets in brain disorders associated with decreased GABA activity. There have been several reports of development of GABA modulators (GABA Receptors, GABA transporters and GABAergic enzyme inhibitors) in the past decade. Therefore, the focus of the present review is to provide an overview on various design strategies and synthetic approaches towards developing GABA modulators. Furthermore, mechanistic insights, structure–activity relationships (SAR) and molecular modeling inputs for the biologically active derivatives have also been discussed. Summary of the advances made over the past few years in the clinical translation and development of GABA receptor modulators is also provided. This compilation will be of great interest to the researchers working in the field of neuroscience. From the light of detailed literature, it can be concluded that the numerous molecules have displayed significant results and their promising potential, clearly places them ahead as potential future drug candidates.

Graphical Abstract

1. Introduction

Gamma aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system (CNS) that exerts various physiological functions by binding at pre- and postsynaptic receptors (GABA receptors) at inhibitory synapses in the brain [1–2]. It is an essential component for the overall balance between neuronal excitation and inhibition that play a vital role in performing normal brain function. Low levels of GABA in the brain have been associated with diseases like anxiety, pain, mania, depression, and seizures, mainly due to overstimulation of other neurotransmitter receptors, transporters, and metabolic enzymes [3]. Treatment of disorders resulting from low levels of GABA involves increasing its concentration by either stimulating its biosynthesis or inhibiting its metabolism. Mammalian GABA receptors can be classified into two major categories: the ionotropic receptors, which are ligand-gated ion channels (GABA_A, GABA_C); and G-protein coupled or metabotropic receptors (GABA_B) [4]. These mammalian GABA receptors are widely distributed in the nervous systems. Metabotropic receptors, upon activation, regulate the formation of IP₃ (second messenger) which inturn elevates the release of Ca²⁺ from intracellular Ca²⁺ channels [5]. It has been revealed that a total of 19 related GABA receptor subunits are distributed in the mammalian nervous system viz. 6α, 3β, 3γ, 1δ, 1Є, 1π, 1θ, and 3ρ [6–8]. Innovative molecular biology has concluded that GABA_A receptors comprise of a pentameric assembly of heteromeric subunits in a 2:2:1 stoichiometry with the most common protein assemblies being α1β2γ2, α2β3γ2, α3β3γ2 and α5β3γ2 [9]. (Fig. 1) In recent past, a homology model of α1β2γ2 GABAA receptors were introduced which lightened the major features of GABA_A receptors and give detailed of mechanism of stabilizing the closed conformation and the interaction of chloride ion with their specific channel pore.[10] Due to their involvement in several neurological and psychiatric disorders, GABA_A receptors as therapeutic targets have played an important role in central transmission processes [10] delivering a large number of anxiolytics, anti-epileptics, sedatives and hypnotics among which Benzodiazepines (BDZ) are the most common ones. (Fig. 1) [11].

Fig. (1).

GABA_A receptor subtypes and their functions

Pharmacological and genetic studies have revealed interesting facts about these reported clinical effects. GABA_A receptors contains the three major drug sites: (i) the picrotoxin sites (channel blocker, NAM), (ii) the GABA sites (agonist/antagonist) in the extracellular domain, (iii) and the BZD sites in extracellular domain. Studies also showed that GABA_A α1 agonism (positive allosteric modulation, PAM) induces sedation, while GABA_A α2 and/or GABA_A α3 agonism imposes the desired anxiolytic effect. Conversely, GABA_A α1 inverse agonism (negative allosteric modulation, NAM) is responsible for pro-convulsant and GABA_A α2 and/or GABA_A α3 inverse agonism (NAM) produces an anxiogenic response [12,13]. The agonistic effect at the GABA_A α5 receptor subtype induces memory impairment which is also a known clinical side effect with the use of Benzodiazepines (BZD) [11]. GABA_A receptors have multiple allosteric modulatory sites for barbiturates, anesthetics, steroid, and BDZ which modulates the opening of the channel through different mechanisms of action [14]. Moreover, non-BDZ ligands have also been discovered to bind within the BDZ site (at the interface of α and γ subunit) e.g imidazopyridines and pyrazolopyrimidines [14–17].

A number of the GABA_A receptor modulators have been reported so far which include Diazepam, Alprazolam, Etomidate, Glutethimide etc. (Fig. 2). These are highly efficacious for cognitive disorders but their reported adverse effects such as sedation, amnesia, ataxia, and abuse liability due to their negative allosteric modulation have limited their clinical utility [19,20].

Fig. (2).

Drugs acting on GABA receptors and GAT inhibitors

Structurally, GABA_B receptors are composed of two heterodimeric subunits: GB1 and GB2 [27]. The GB1 subunit has been shown to bind GABA but is unable to activate G-proteins [28]. Whereas GB2 can activate G-proteins but cannot binds directly to GABA [29]. Functionally, GABA_B receptors mediate slow synaptic inhibition and are involved in numerous types of nociception, cognitive impairment, epilepsy, spasticity, and drug addiction [30].

The next important class of receptors is GABA_C receptors which belong to homomeric ligand-gated ion channel [31], are insensitive to bicuculline (GABA response blocker), BZD, neurosteroids, and sedatives [32–34] These receptor subtypes are very limited in distribution throughout the central nervous system which is the main reason for reduced risk of potential side effects when targeting these receptors [35]. They are generally expressed within retina [36], hippocampus [37,38] and superior colliculus (SC) [39]. A recent investigation of GABA_C receptor revealed that its antagonism enhances cognition and memory [40], however, there is no report on approved GABA_C receptor modulators being used clinically in the treatment of memory deficit and cognition. Although, clinical trials involving GABA_C antagonist 3-aminopropyl(butyl)phosphinic acid (SGS742, Fig. 2) show cognition and memory enhancement [40] however, this drug is not selective for GABA_C receptors. These findings highlight the pressing need to develop more selective, and active GABA_C receptor antagonists to determine the role of GABA_C receptors in cognitive processes.

Besides these receptor subtypes, various GABA transporters (GAT) also play a vital role in GABAergic neurotransmission. Four types of GABA transporters are known- GAT-1, GAT-2, GAT-3 and BGT-1 [41–48] are found in humans. Amongst these, GAT-1 and GAT-3 have been shown to be predominantly responsible for the transport of GABA into neurons and glial cells, respectively, indicating that GAT-1 and GAT-3 transmembrane proteins are located almost exclusively in the detectable amounts [50,51]. Hence only GAT-1 and GAT-3 are of special interest when it comes to the development of CNS drugs. Examples of selective GAT-1 (SK & 80076A and tiagabine) and GAT-3 (SNAP-5114) inhibitors are mentioned in Fig. 2. Exploring the Steered molecular dynamics (SMD) technique, in which harmonic restraining potential is applied to ligand and their dissociation and re-association occur, which is further used in determining the substrate (GABA) translocation and inhibitor (tigabine) mechanism of action.

In view of the prominent role of GABA receptors and GABA transporters in various neurological functions, this review highlights the discovery of GABA analogs and GAT inhibitors in the last decade. A detailed description of the design strategies for the synthesis and structure-activity relationships (SAR) revealed during the biological evaluation of GABA modulators, the binding interactions of molecules with the GABA receptor subtypes as well as drugs that have gone into clinical trials has been presented in this review. We believe, that this compilation will be of great interest to the researchers working in the field of neuroscience drug discovery. In the present review, various GABA modulators have been categorized according to their pharmacological aspects as follows:

1. GABA receptor modulators

   1. GABA_A receptor modulators

      1. O-Heterocycles

         1. Pyran derivatives
         2. Miscellaneous
      2. N-Heterocycles

         1. Pyrazole derivatives
         2. Quinoline & Quinazoline derivatives
         3. Pyridine derivatives
         4. Imidazole derivatives
         5. Pyridazine derivatives
         6. Pyrimidine derivatives
      3. N & O-Heterocycles

         1. Isoxazole derivatives
      4. N & S-Heterocycles

         1. Thiazole derivatives
      5. Miscellaneous derivatives
   2. GABA_B receptor modulators
   3. GABA_C receptor modulators

2. GABA transporter (GAT) or GABA uptake inhibitors

3. GABAergic enzyme inhibitors

2. Rational approaches towards the design of GABA receptor modulators, transporters and metabolic enzyme inhibitors

2.1. GABA receptor modulators

2.1.1. GABA_A receptor modulators

(a). O-Heterocycles

(i). Pyran derivatives

Prompted by various pharmacological attributes of flavylium compounds such as anti-mutagenic [52], anti-tumoral [53], or anti-malarial activities [54] Stotz et al. synthesized various substituted flavylium derivatives and evaluated their binding affinity towards the Benzodiazepines binding site of GABA_A receptor from rat brain membrane by determining the displacement of [³H]-flumazenil. The nanomolar range affinity of these flavylium cations was explained on the basis of their chemical transformation into corresponding trans- retrochalcone. In-vitro studies revealed that compound 1 (trans- retrochalcone) was more active than its corresponding flavylium derivatives i.e. compounds 2 and 3 showed Ki values 280 nM, 60 nM, and 140 nM respectively (Fig. 3) [55].

Fig. (3).

Flavylium derivatives as GABA_A receptor modulators

Biologically inert (‘caged’) carboxyl-linked GABA precursors (4) as well as the 7-(N,N-diethylamino)-4-(hydroxymethyl)coumarin (DECM) moiety release free GABA by a photoactivation mechanism without hindering the biological system and thus can be used to investigate the mechanism of GABA_A receptor function [56,57,58] Inspired by these findings, L. Fan et al. described a new series of carboxy- substituted DECM-GABA derivatives. Among all the synthetics, only the caged molecule 5 was active and released GABA, triggering the GABA_A receptors channel opening in the cell membrane. As compared to other developed molecules, it easily released GABA with a quantum yield of 0.1 at 400 nM. These findings indicated that caged 5 could be used in transient kinetic measurements of GABA_A neurotransmitter receptors on cell surfaces without causing damage to key cellular constituents (Fig. 4) [60].

Fig. (4).

Coumarin derivatives as GABA_A receptor modulators

K. N. Mewett et al. synthesized a series of flavan-3-ol derivatives as positive modulators of GABA_A α₁β₂γ_2L receptors. All the synthetics were evaluated for their ability to potentiate the GABA response, by using human recombinant α₁β₂γ_2L GABA_A receptors expressed in Xenopus laevis oocytes. A well-established structure-activity relationship, summarized in Fig. 5, was obtained from the results which revealed that the trans-(2S,3R)-isomers of 6 and 7 were more active than their corresponding cis isomers [61,62]. Compounds 6 and 7 were found to exhibit a significant positive modulatory effect on GABA_A receptors with EC₅₀ values of 6.68 and 12.15 μM respectively and could be used for further CNS investigations (Fig. 5) [63].

Fig. (5).

Flavanol derivatives as GABA_A receptor modulators

To characterize the phytochemistry of lipophilic extract of Hypericum lissophloeus (Hypericaceae), Crockett et al. isolated and identified new chromanone derivative that potentiated GABA_A current. Compound 8 (5,7-dihydroxy-2,3-dimethyl-6-(3-methyl-but-2-enyl)-chroman-4-one), was tested for its ability to potentiate the GABA current at recombinant α₁β₂γ₂ GABA_A receptors present in Xenopus oocytes by using two-electrode voltage clamp method. It showed maximal potentiation with EC₅₀ and I_GABA-max values 4.5 ± 2.6 μM and 4431 ± 478 % respectively. Testing compound 8 on different GABA_A subtype receptors revealed that maximal potentiation was dependent mainly on the type of α_, β and γ subunits combination present in the subtype receptors. (Fig. 6) [64].

Fig. (6).

O-Heterocycles as GABA_A receptor modulators

(ii). Miscellaneous

Previously studied caged bicyclophosphates (acting as convulsant) showed toxicity in mice model likely due to bridgehead substitution with tert-butyl group or due to inhibition of GABA mediated synaptic transmission [65,66]. These observations led to the synthesis of tert-butylbicyclophosphorothionates (TBPS: 9) in the ³⁵S-labelled form, used for studying vertebrate GABA_A receptors [67,68]. There was an increase in the insecticidal activity against housefly GABACls by the introduction of an alkyl group to the endocyclic methylene of bicyclophosphorothionates. However, there was no Electrophysiological evidence which showed that these modified bicyclophosphorothionates act against insects’ GABACls [69,70]. Akiyoshi et al. provided this evidence by studying 4-isobutyl-3-isopropylbicyclo-phosphorothionate as a selective blocker of insect GABA-gated chloride channels. Invertebrate GABACls and glutamate-gated chloride channels (GluCls) (inhibitory neurotransmitter receptors) are important targets for insecticides and anthelmintics. To determine the mechanism of 10, GABA and glutamate concentration-response relationship in the presence and absence of 10 at IC₅₀s in P.americana neurons were examined. The result of GABA-concentration-response curve (showed a reduction in I_max without significant change in EC₅₀ indicating that 10 act as a non-competitive antagonist in both P.americana GABACls and GluCls. On the other hand, the results of [³H]EBOB (4-ethynyl-4-n-propylbicycloorthobenzoate) binding assay showed that 10 was more potent in insects GABACls than mammalian GABACls with their IC₅₀ values 2.36 μM and 45.2 μM respectively. Furthermore, site-directed mutagenesis studies of GABA_A receptors containing α₁ and β₃ subunits indicated that the TBPS-binding sites reside within the pore of channel and speculated that compound 9 with a tert-butyl group, may fit into a small channel pore created by large hydrophobic 2’side chains whereas compound 10, with an isobutyl and isopropyl group, may fit well into a large hydrophobic pore created by 5-methyl side chain of 2’ alanines (Fig. 6). Moreover, to understand the binding mode of benzofuran and their antianxiety potential Yoval A et al. synthesized the 2,3-disubstituted Benzofuran Analogues of GABA act as neurotropic agents. All the synthetics were tested for their neuroleptic activity on a mouse model of seizures that induced by pentylenetetrazol (PTZ) or on 4-aminopyridine. The results indicated that compound 11 showed significant anxiolytic activity[71].

(b). N-Heterocycles

(i). Pyrazole derivatives

In order to increase the potency and interactions with the receptor protein (GABA) Guerrini et al. carried out structural modifications at 3^rd position of a previously reported compound 3-(3-thienyl)-8-chloropyrazolo [5,1-c][1,2,4]benzotriazine 5-oxide (12) [72] and synthesized new derivatives of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine. These synthesized compounds were strictly related to indiplon [73,74] and ocinaplon [75] which have been reported for their anxiolytic activity through GABA_A/Benzodiazepine receptor complex [76–80]. All synthesized compounds were evaluated for their binding affinity at GABA_A/Benzodiazepine receptor complex by displacing [³H] flumazenil (R_O15–1788). Five other parameters i.e. anticonvulsant activity (using pentylenetetrazole-induced convulsant), motor coordination activity (by rota-rod test), potential anxiolytic activity (by using light/dark choice test), spontaneous motility and explorative activity (with hole board test) were also considered for evaluation. Binding studies revealed that compounds having a five-membered heteroaryl group (compound 13, 14), showed strong affinity with Ki values of 28 nM, 8.9 nM, respectively. Anticonvulsant activity was studied in mice using pentylenetetrazole (PTZ) as chemical convulsant agent and latency time was measured. These results showed that only compound 13 showed potent activity at the dose of 3 mg/kg with latency time 328.5 ± 38.0 s. Moreover, spontaneous motility and explorative activity were tested by Hole-board test method and the results revealed that compounds 13 and 14 were able to diminish curiosity at the oral dose of 3 mg/kg and 30 mg/kg. Anxiolytic activity was also studied by using dark/light box apparatus and where compound 14 showed potent activity at oral doses of 10 and 30 mg/kg as compared to diazepam. The results of motor coordination were evaluated by rota-rod apparatus and results indicated that none of the newly synthesized compounds induced any effect in mice. Molecular docking study of the most potent compound 13 was also performed to access the preferred geometry of protein-ligand complex (Fig. 7) [81].

Fig. (7).

3-acylpyrazolo[5,1-c][1,2,4]benzotriazine derivatives as GABA_A receptor modulators

Previous studies revealed that indiplon (15) selectively bound to the GABA_A-α₁ receptors and showed binding affinity at rat cerebellar and cerebral cortex membrane in nanomolar ranges with Ki values of 0.55 and 0.45 nM respectively [73]. Prompted by these findings, Hoepping et al. synthesized a series of fluorinated analogs of pyrazolopyrimidines (due to superior imaging properties of ¹⁸F than ¹¹C) by various structural modifications of the lead compound indiplon [82]. Synthesized compounds were evaluated in-vitro for their binding affinity at GABA_A receptor complex. Compound 16 showed greater affinity at cerebellar GABA_A receptors with an IC₅₀ value of 2.78 ± 0.63 nM comparable to the lead compound indiplon (IC₅₀ 3.29 ± 0.37 nM). The SAR study revealed that by replacing the methyl group with a fluorinated alkyl group (i.e. fluoropropyl group) showed potent binding affinity. Lack of carbonyl group drastically decreases the binding affinity. By replacing thiophene ring with fluorophenyl ring leads to a decrease in binding affinity whereas by replacing acetyl group with 4-fluorobenzoyl and 4-nitro benzoyl moiety compounds sustained the binding affinity towards GABA receptors. Moreover, to check the ability of synthesized compounds to cross the blood-brain barrier, log P values were determined (0.9–2.5) which indicated a good brain penetration. Log D values were found in the range between 2.2 and 2.7 which showed that the newly synthesized compounds were able to cross the blood-brain barrier sufficiently (Fig. 8) [83].

Fig. (8).

Pyrazolopyrimidine derivatives as GABA_A receptor modulators

Inspired by previously reported pyrazolo[5,1-c][1,2,4]benzotriazine-5-oxide compounds that possessed anxiolytic-like properties without exhibiting sedative and myorelaxant effects [84–86,81], Guerrini et al. synthesized new 3-methyl- and 3-iodo-, 8-aryalkyloxy and 8-arylalkyl aminopyrazolo[5,1-c][1,2,4] benzotriazine derivatives. The Benzodiazepine site/GABA_A receptors binding affinity of these newly synthesized compounds was evaluated by determining their ability to displace [³H] flumazenil from its specific binding in bovine membrane. All compounds exhibited significant binding, amongst which compounds 17, 18, 19 (5-N-dioxide form of 13) and 20 showed higher potencies with their Ki values in the subnanomolar range (0.78, 0.109, 0.71 and 0.138 nM respectively). These binding studies helped establish a SAR which is summarized in Fig 9. Furthermore, to check the anti-anxiety effect, in vivo studies were carried out by using male Swiss albino mice (23–30 g) and male Sprague Dawley rats by employing the light and dark model. Compound 18 (10 mg/kg po) showed significant anxiolytic effect, compared to diazepam (taken as reference compound). Animals were further tested for pain reduction by using paw-pressure test where compound 18 was found to exhibit antihyperalgesic effect potently with 3–30mg/kg po dose. Moreover, antihyperalgesic potential of compound 18 was also evaluated in animal Streptozotocin (STZ)-induced hyperalgesia pain model [87].

Fig. (9).

Aminopyrazolo[5,1-c][1,2,4] benzotriazine derivatives as GABA_A receptor modulators

To identify novel compounds with improved subtype selectivity Hintermann et al. introduced a novel series of an agonist of GABA_A receptors using pyrazolone derivative (CGS-9896 (21)), a BDZ-site ligand [88] as a lead molecule. Compound 21 was reported as anxiolytic but showed less sedative effects in rodents. Modifications on phenyl pyrazolone moiety led to the derivative 22 containing chloro group at 4-position with higher affinity for both GABA_A receptors subtypes, α1 and α2 but with weaker efficacy than that of the parent molecule. Results indicated that the 4-chloro phenyl ring was crucial for affinity with the receptor subtype. Keeping 4-chloro phenyl ring intact, quinolone core structure was then modified to compound 23 with higher activity (IC₅₀ = 271 nM (α2). Furthermore, to improve the potency and subtype selectivity, the 2-chlorophenyl ring was modified. Results indicated that placing a chloro-substituent on different positions was detrimental for selectivity whereas replacing the 2-chloro with 2-fluoro substituent exhibited very promising functional selectivity for the a2 subtype. Trifluoro-methyl substituent possessed maximal efficacy without any selectivity. Similar results were also found with the 4-fluoro analog. The substitution of cyclopropyl substituent led to compound 24 which exhibited higher efficacy towards α1 subtype. The main complication with compound 23 was its high renal clearance in rat model (128.3 μL/min/mg), limiting its further use in animal models. Therefore, in-vitro metabolism study with rat liver microsomes was carried out which recognized two major pathways of compound 23 i.e. extensive hydroxylation of aliphatic part of the core (pathway A) and presence of significant amount of parent pyrazolone (pathway B). Based on these particular findings, new series of compounds were synthesized with the aim to lower the in-vitro clearance and to increase the metabolic stability. Modifications were carried out on the tetrahydroquinoline core and results stated that by the introduction of an oxygen or methyl-substituent at the 8-position led to decreased core-hydroxylation. Moreover, replacement of benzyl by phenyl ring resulted to largely improved stability. Replacing the 2-chloro-benzyl group with the cyclopropylmethyl group resulted in better functional selectivity for a2. Finally, incorporation of N-imidazole and cyclopropyl group into the skeleton (25), resulted in desired as well as interesting results. Compound 25 among this series showed improved stability and high specificity for both GABA_A receptor. These results made compound 25 a lead drug candidate for the further development of potent and selective GABA_A receptor modulators (Fig. 10) [89].

Fig. (10).

Pyrazolone derivative as GABA_A receptor modulators

In continuation, [85] Guerrini et al. designed and synthesized new series of derivatives containing a pyrazolo[5,1-c][1,2,4]benzotriazine by using compound 26 as lead molecule (3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine-5-oxide) and identified ligands which bind to the Benzodiazepine (BDZ) site on the GABA_A receptors endowed with the potential of enhancing cognition activity without producing any side effects, usually associated with non-selective GABA_A modulators [80]. All new compounds were studied in-vitro and in-vivo for their potential effect on learning, memory, and anxiety. The BZ site/GABA_A binding affinity (Ki) of newly synthesized compounds was evaluated by their ability to displace [³H]flumazenil (Ro15–1788) from its specific binding in bovine brain membrane. Furthermore, Light/Dark box and Rota rod apparatus were used to check the anxiety effect and motor incoordination activity by using a mouse model. The compounds 27-30 showed excellent binding affinity as compared to lead molecule by replacing O atom with the –NH- group at position 8 with their Ki values 0.33 ± 0.08 nM, 0.33 ± 0.07 nM, 0.72 ± 0.06 nM, 0.23 ± 0.02 nM and 1.30 ± 0.10 nM respectively, whereas compound 28 showed recombinant subtype affinity to bind at α₁ and α₅ subtype receptors with Ki value 0.44 nM and showed % age inhibition (I% = 37) to α₂ subtype receptors. Pharmacological data indicates that the compounds 31, 32, 33 and 34 act as dual functional modulators of GABA_A receptors (pro-mnemonic and anxiolytic agents) in the range of 10–30 mg/kg while compounds 28 and 35 displayed good antiamnesic and precognitive activity at 1 and 3 mg/kg. Structure-activity relationship showed that the isosteric substitution of O/NH (e.g compound 27) showed excellent binding affinity, which is also maintained in 3-and 2- pyridyl derivatives indicating that the NH group is crucial for receptor recognition. Furthermore, by replacing the pyridine ring with five-membered heterocycles showed positive results (Fig. 11) [90].

Fig. (11).

Pyrazolo[5,1-c][1,2,4]benzotriazine derivatives as GABA_A receptor modulators

(ii). Quinoline & Quinazoline derivatives

Previously it was reported that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate (36) and 3-carboxamide (37) derivatives showed binding towards Benzodiazepine sites. A pharmacophore model of the binding site was thus developed to identify and optimize novel 4-quinolones derivatives of 36 and 37 [91,92] which were expected to be hydrolyzed rapidly by ester/amide hydrolysis. Based on these particular findings, Lager et al. synthesized new 3-acyl substituted quinolones derivatives to 1) study the effect of acyl substituent in position 3, instead of a carboxylate or carboxamide, by preparing more stable ketone analogs of 36 and 37; 2) Evaluate the subtype selectivity of 4-quinolones; and 3) analyze the pharmacophoric region. All the synthetics were evaluated for their binding affinity determined by displacing ³H-Flumazenil from their binding sites and also assess their specificity by using recombinant α₁β₂γ_2s, α₂β₂γ_2s, and α₃β₃γ_2s receptors subtypes. It was shown that all the synthesized compounds exhibited comparable activity to previously reported alkyl substituted carboxylate and carboxamide quinolone derivatives. Compounds 38–41 displayed strong binding affinity towards the receptor α₁β₂γ_2s as well as showed higher α₃/ α₁ Ki ratio values (except 38 and 40), as compared to carboxylate group respectively. Structure-activity relationships described that benzyl group at 6^th position (R₂) enhances the binding affinity three times more as compared to the ethyl substitution (Fig. 12) [93].

Fig. (12).

3-acyl quinolones derivatives as GABA_A receptor modulators

Nilsson et al. synthesized a series of azaflavone derivatives and tested for their affinity towards Benzodiazepines binding sites of recombinant α₁β₃γ_2s, α₂β₃γ_2s, α₄β₃γ_2s, α₅β₃γ_2s GABA receptors by measuring their ability to displace ³H-flumazenil from HEK293 cells [94]. It was concluded that azaflavones were less active as compared to their corresponding flavones. However, 42 and 43 exhibited potent binding affinity and activity. Valuable information was obtained from the binding mode of the azaflavones and flavones which revealed that flavones only interact as hydrogen bond acceptors with hydrogen bond donor site in the binding pocket. Aza-flavones, for example, compounds 42 and 43, could lead as a hydrogen bond acceptor and donor by interacting with A3 (Hydrogen bond accepting site). In azaflavones, the substitution of the 5-benzyl group at arylquinolones in place of 5-Bromo, showed five times higher binding affinity and ratio values towards α₂β₃γ_2s, α₃β₃γ_2s GABA_A receptors (Fig. 13) [95].

Fig. (13).

Azaflavoves as GABA_A receptor modulators

To combine subtype selectivity of both modulators i.e. positive allosteric modulators (PAM) acting on α₂ and α₃ and negative allosteric modulators (NAM) acting on α₅ receptors, Liu et al. synthesized two new series of non-Benzodiazepines derivatives (cinnoline and quinolone) acting as dual-functional modulators at GABA_A receptors. Synthetics were evaluated by using oocyte two-electrode voltage clamp model. The binding affinity of these synthetics was also evaluated by determining the displacement of non-selective Benzodiazepines site modulator [³H]flunitrazepam from the binding sites. The results of NAM screening showed that compound 44 (quinoline derivative) was ‘neutral antagonist’ at α₁-GABA_A receptors, ‘partial agonist’ at α₂-/α₃-GABA_A receptors and ‘partial inverse agonist’ at α₅- GABA_A receptors as compared to diazepam and DMCM (methyl-6,7-dimethoxy-4-ethyl-β- carboline-3-carboxylate) with Ki values 14 nM and 8.3 nM for α₅- GABA_A and α₂-GABA_A receptors respectively. On the other hand, compound 45, containing cinnoline nucleus showed efficacy towards α₅- GABA_A receptor subtype. Furthermore SAR studies revealed that the pyrimidine side group at the 5-position of quinoline and 8-position of cinnoline was crucial for the NAM_S activity at α₅- GABA_A receptors and also the position of the two nitrogen atoms in 2-pyrimidyl ring in cinnoline and quinolone core structure may impact the compounds’ efficacy as α₅- GABA_A receptors NAM_S (Fig. 14) [96].

Fig. (14).

Cinnoline and Quinolone derivatives as GABA_A receptor modulators

Quinoline (46) and cinnoline (47) are two reported GABA_A receptor modulators exhibiting α1 PAM (anxiogenesis) effect and moderate α2 and α3 PAM (anxiolytic) effect with high human microsomal clearance [97,98]. These pharmacological findings prompted Alhambra et al. to synthesize their derivatives by making stepwise modifications in the parent skeletons. Oocyte voltage clamp study was used to evaluate all the derivatives. In an attempt to diminish the PAM effect of α1, methyl substituted phenyl ring was placed at the 8^th position of cinnoline. Resulting compounds showed inverse agonistic effect (NAM) on α1 GABA_A receptor. Positive results were also found for α2 and α3 PAM efficacy with two methyl groups, specifically 2,5-dimethyl, on the 8-phenyl ring whereas the aqueous solubility of most of the synthetics was very low. Keeping in mind the low solubility, compounds were modified by incorporating oxygen atom in between the methyl group and phenyl ring which led to the compounds with at least 4-fold enhanced aqueous solubility than the parent molecules. Simultaneously, the amide group of cinnoline was substituted but it failed to the desired results as the substitution was only sensitive towards the modulation of α1 GABA_A receptor. From the positive functional modulatory activity on GABA_A receptors resulting from methoxy substituted phenyl ring on cinnoline, quinolone nucleus was then modified using 2,5-dimethoxy phenyl ring at the 5^th position of quinolone and alkyl substitutions on Nitrogen of beta-lactam ring. Most compounds exhibited interesting α2 and α3 PAM activity and better α1 NAM activity. N-cyclobutyl and N-cyclopropyl moieties were found crucial for α1 NAM activity. Among these series synthesized, none of the compounds showed higher specificity for α2 and α3 modulation activity. Next, fluorine was introduced at the 6^th position of quinolone and 7^th position of cinnoline which resulted in slight enhancement in functional activity. Furthermore, the 5- and 8-phenyl ring on both quinolone and cinnoline nucleus were substituted with simple alkyl, alkoxy, and heteroaromatic substitutions. Interesting results were obtained by this modification which indicated that a) methoxy group ortho to the biphenyl linkage of both 46 and 47 provides beneficial results, b) heterocycles and nitrogen at the 3-position relative to the biphenyl linkage also imparts the desired functional activity profile and, c) a fluoro group ortho to the biphenyl linkage helped provide the desired profile in one instance. From these findings, two compounds 46 and 49 (AZD7325) were found as most potent with desired activity profile with solubility 3.9 and 7.6 μM. The IUPAC name of AZD7325 is 4-amino-8-(2-fluoro-6-methoxy-phenyl)-N-propylcinnoline-3-carboxamide. These two derivatives have been used as clinical candidates to treat the complications related to GABA_A receptors (Fig. 15) [99].

Fig. (15).

Quinolone and Cinnoline analogs as GABA_A receptor modulators

Moran et al. reported the radiosynthesis of a potential radiotracer by radiolabeling the two quinolones i.e. [¹¹C]9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one ([¹¹C]50) [10] and [¹¹C]9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one ([¹¹C]51) because 1) they showed potent activity for GABA_A α₁ and α₂-subunits but were selective only for α₅- subunit which was reported on the basis of structure-activity relationship studies [99] 2) they are susceptible for radiolabeling at methoxy position 3) these selective compounds represent a new class of quinoline-based radiotracers for imaging the BZ site of GABA_A receptors [99,100]. Both radiotracers 50 and 51 were evaluated ex vivo in rat brain region and whole blood whereas radiotracer 52 aws evaluated for its ex vivo brain distribution studies following tail vein injection in conscious rats where it low level of radioactivity (< 0.2% ID/g). Preliminary bio-distribution studies demonstrated that [¹¹C]51 readily crossed the blood-brain barrier and had an appropriate brain distribution for GABA_A receptors. Furthermore, compound 51 also display selective binding to GABA_A receptors (Fig. 16) [101].

Fig. (16).

Carbon-11 labelled radiotracers as GABA_A receptor modulators

Prompted by the significant insecticidal property of naturally occurring plant alkaloid Alantrypinone (+53) [102] (polycyclic alkaloid isolated from Penicillium thymicola), and to understand its interaction with GABA receptors, T. Watanabe et al. synthesized a series of alantrypinone derivatives having significant insecticidal properties with the selective antagonistic activity of GABA receptors. Prior to examining the potency of natural alkaloid (+53) and its synthetic derivatives, GABA receptor antagonistic effect of (+53) was evaluated by whole-cell patch clamp method using American cockroach (Periplaneta americana L.) neurons. Results showed that the (+53) acts as an antagonist at insect GABA receptors. Furthermore, the insecticidal potential was also tested by employing housefly GABA receptors (Musca domestica L.) using the high-affinity non-competitive GABA receptor antagonist [³H]EBOB. The compound (+53, exo) exhibited higher inhibitory potency than its racemate (±53, endo). Compound ±53 and its synthetic analog ±54, were found to be the most potent ones exhibiting almost complete inhibition of [3H] EBOB binding at 10 μM concentration. It concluded that methyl and isopropyl substitutions play a significant role for activity. Furthermore, docking study of most potent compound 54 with the human β3 GABA receptor presented that larger substituents could not fit the channel pore binding site and also corroborated the amide group present at 2^nd and 18^th position bring crucial for high activity. (Fig. 17) [103].

Fig. (17).

Alantrypinone derivatives as GABA_A receptor modulators

(iii). Pyridine derivatives

Inspired by previous work [104–107], Peterson et al. synthesized a novel series of GABA_A receptor agonists containing 2-aminopyridine or guanidine /amidine ring system analogs as novel amine bioesters which challenge the restricted structural diversity of GABA_AR and to make easy for the search of subtype selective GABA_AR agonists. All the synthetics were tested for their binding affinities at GABA_AR by determining the displacement of [³H] muscimol in rat membrane and characterization was carried at human α₁β₂γ_2s and ρ1 GABA_ARs expressed in tsA201 cells using FLIPR™ (FMP assay) membrane potential blue assay. The results of the binding assay showed that compounds containing carboxylic acid and acetic acid moieties at 4 and 5 positions (55-58) and tetrahydropyridine analogues of compound 55 displayed good binding affinities as compared to the reference compounds (GABA, Muscimol, Isoguvacine, Isonipecotic acid). In the FMP assay, compounds 59 and 60 act as partial agonist at the α₁β₂γ_{2s and} ρ1 GABA_ARs whereas full agonist at α₅β₂γ_2s as compared to the reference compounds. From the docking studies, it was revealed htat compound 55 showed the same binding mode as GABA and muscimol including hydrogen bonding interactions between the protonated amidine group and β₂Glu155 and carbonyls of β₂Ser156 and to β₂Tyr157. The carboxylic acid group of the molecule and Arg66 showed alternative orientations (Fig. 18) [108].

Fig. (18).

2-aminopyridine or guanidine /amidine analogs as GABA_A receptor modulators

(iv). Imidazole derivatives

Benzodiazepines show memory impairment as an undesired clinical result most probably due to their agonistic effect at the GABA_A α5 receptors subtype and medicament congeners with inverse agonistic effect could treat memory impairment [11]. Developers from Merck discovered an efficient GABA_A α5 receptor inverse agonist (61) which could treat alcohol-induced memory impairment, however, it was withdrawn due to its severe side effects like, high precipitation, crystalluria and nephrotoxicity [109,110]. From these findings, G. Achermann et al. evaluated various series of imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]-Benzodiazepines to develop effective and selective inverse agonists for GABA_A α5 receptor subtype. Cloned rat or human receptor subunits were expressed in insect SF9 cells, human HEK293 cells or Xenopus laevis oocytes for 3H-Flumazenil GABA_A α1–5 binding or electrophysiology. Efficacy was determined as the percentage change of a submaximal response to GABA. Lead compound 62 displayed good affinity as well selectivity towards GABA_A α5 receptor but was found to act as an agonist on the same. Further modifications on compound 62 resulted in compound 63 which showed an increase in affinity, selectivity and inverse agonistic activity on GABA_A α5. Structure-activity relationship revealed some interesting facts about the selection of groups on the molecular skeleton: a) replacement of ester group (at R₃) with an electronegative atom exhibited loss of affinity b) –Cl atom in place of ester was found to be suitable for potency but was not able to show inverse agonistic effect for GABA_A α5 receptor subtype c) introduction of acetylene group in place of the electronegative atom at R₁ further increased inverse agonistic effect and selectivity for α5 as in compound 64. Furthermore, keeping intact the imidazobenzodiazepine scaffold the effect of various five-membered heterocyclic rings was tested where compound 65 related to imidazo[1,5-a]pyrazolo[1,5-d][1,4]-Benzodiazepine class delivered very potent GABA_A a5 receptor subtype inverse agonistic effect. Among these series Diimidazo[1,5-a:1’,2’-d][1,4]-Benzodiazepine was found to exhibit promising results (higher affinity, selectivity and efficacy). Unfortunately, pharmacological profile explained its relatively high microsomal clearance which was the main drawback of this series. Imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]-Benzodiazepine was a series with interesting results showing moderate affinity and selectivity but higher efficacy among which compounds 65 and 66 were found to be endowed with most prominent results with best pharmacological properties. From the overall study, it has been concluded that Imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]-Benzodiazepine series could be used for further lead optimization as CNS acting agents (Fig. 19) [12].

Fig. (19).

Imidazobenzodiazepine derivatives as GABA_A receptor modulators

(v). Pyridazine derivatives

To enhance the GABA_A receptor antagonistic activity of Gabazine 67 (para-methoxy analog), Iqbal et al. carried out the structural modification of gabazine to synthesize a novel series of arylpyridazine analogs. The synthesized compounds were examined for their potency to antagonize GABA_A receptors by using patch clamp electrophysiology with recombinant α1β2γ2s GABA_A receptors in HEK293 cells. Concentration-inhibition curves obtained from the study revealed some interesting facts about the structure-activity relationship which stated that compound 68 with benzyl substitution as R, showed significant increase in potency (IC₅₀ = 11 nM) as compared to gabazine (IC₅₀ = 349 nM). Introduction of meta-methoxy benzyl group as in 69, further potentiated the antagonistic activity with an IC₅₀ value of 7 nM. Introduction of an electron withdrawing nitro-group at the meta-position of benzyl ring (70) was well tolerated (IC₅₀ = 3 nM) as compared to 69. Furthermore, to check an alternative substitution pattern, propargyl group (71) in the skeleton of gabazine was made which also exhibited potent inhibition as compare to gabazine with IC₅₀ value 40 nM (Fig. 20) [111].

Fig. (20).

Arylpyridazine analogs as GABA_A receptor modulators

Prompted by the insecticidal property of Gabazine [112–114] (pyridazine derivative), Rahman et al. synthesized 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acid derivatives and evaluated for their insecticidal potential via antagonism of GABA receptors by using fluorometric imaging plate reader (FLIPR) membrane potential (FMP) assays against small brown plant hopper, common cutworm, and American cockroach GABA receptors. The antagonistic potential of the compounds was determined by fluorescence reduction induced by the synthesized compounds. Most of the compounds exhibited moderate to good results among which 3,4-methylenedioxyphenyl and the 2-naphthyl congeners (72 and 73) inhibited small brown plant hopper (SBP) and common cutworm (CC) GABA receptors almost completely at a concentration of 100 μM while the compound consisting of 4-biphenyl moiety (74) showed complete inhibition of American cockroach (AC) GABA receptors when tested in patch-clamp technique. Furthermore, the type of inhibition of GABA_A receptors was also checked which presented that the compounds inhibited receptors competitively at 500 μM concentration. The binding interactions with the GABA_A receptors (housefly) of representative compound 74 was also rationalized by using molecular modeling studies. 74 was stabilized within the orthosteric active site of GABA receptor by CH/π-interactions by its large substituent at 3^rd position (Fig. 21) [115].

Fig. (21).

4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acid derivatives as GABA_A receptor modulators

(vi). Pyrimidine derivatives

Faisi et al. designed and synthesized 1,2,4-triazolo [1,5-α]pyrimidinone and 3-amino-1,2,4-triazole derivatives based on the pharmacophore model of Benzodiazepines (BDZ) binding sites of GABA_A receptors. Conformational analysis was carried out to check whether the synthesized compounds were able to inhibit the structure of BDZ agonist by superimposing energy minima conformers of synthesized compounds on estazolam. All the synthetics were evaluated for their in-vivo affinity towards BDZ receptor using radioligand receptors binding assay. Moreover, the most potent compound (75) was further studied for in-vivo pharmacological effects and molecular docking study was carried out using the BDZ binding sites of GABA_A receptors. Compound 75 showed a higher affinity towards central Benzodiazepines receptors (CBR) than Diazepam (CBR agonist) with a Ki value of 0.42 nM. Results from docking study stated that the carbonyl group of compound 75 is oriented in the same directions as that of the nitrogen moiety of estazolam and thus forms hydrogen bonding interactions with α₁Thr206 and α₁Thr209. Also, the phenyl ring of 75 binds in the lipophilic pocket of γ₂Val190 and γ₂Phe77 formed by the receptor. In-vivo study showed that the compound 75 showed notable hypnotic activity and less anticonvulsant activity without causing any impairment on learning and memory tests in mice. Pharmacological effects of compound 75 were antagonized by flumazenil indicating that the biological effects of these compounds were mainly dependent on the BDZ receptors (Fig. 22) [116].

Fig. (22).

Triazolo [1,5-α]pyrimidinone derivatives as GABA_A receptor modulators

(c). N & O Heterocycles

(i). Oxazole derivatives

To improve the biological profile of previously reported 5-(4-piperidyl)-3-isoxazolol (76; 4-PIOL) derivatives [117] and to develop α subunit selective compounds, Jansen et al. synthesized a new series structurally related to the 4-PIOL by replacing the 3-isoxazol moiety with 1,3,5-oxadiazol-2-ol. All the synthetics were screened for their efficacy to bind to GABA_A receptor by using [³H] muscimol binding assay. Biological results revealed that compound 77 showed higher percentage inhibition of [³H] muscimol binding to the cortex and cerebellar membranes (99 and 97 % respectively at 100 μM) with IC₅₀ values of 1.4 and 1.1 μM respectively. Furthermore, to determine the functional effects on α_iβ₃γ₂ (i =1–6) receptors, patch-clamp experiments with heterologously expressed GABA_A receptors in HEK 293 was carried out. The α-subunit specificity was tested in α_iβ₃γ₂ (i=1–6) GABA receptors by measuring the modulation of GABA induced chloride current and the intrinsic activity of these synthesized compounds. Compound 77 induced currents in all GABA_A receptors with an efficacy greater than 50% as well as possessed higher α subunit specific potency. It was also found that α₆ subunits were most sensitive EC₅₀ 30 μM) whereas α₃ subunits were the least sensitive (EC₅₀ 2300 μM). Interesting results were found in GABA modulatory activity, which showed that compound 77 was mainly responsible for the enhancement of α₅ (423 ± 25%) and α₆ (437 ± 21%) subunits efficacy as compared to α₁ containing receptors. Interesting features about the structure-activity relationship of the synthesized compounds and comparison with the previously reported 4-PIOL derivatives are described in (Fig. 23) [118].

Fig. (23).

5-(4-piperidyl)-3-isoxazolol derivatives as GABA_A receptor modulators

Keeping in view the vast biological applications of isoxazoline, [119] Lahm et al. synthesized 4-heterocyclic aryl isoxazoline to act as potent blockers of GABA gated chloride channel with lack of cyclodiene cross-resistance. All the synthetics were tested for insecticidal potency by using insects such as fall army worm (Spodoptera frugiperda, Sf), corn earworm (Helicoverpa zea, Hz), potato leafhopper (Empoasca fabae, Ef) and western flower thrips (Frankliniella occidentallis, Fo). Among all the synthetics, compounds 78 and 79 containing cyano group showed potent activity with their LC₅₀ values 0.2 ppm for Sf, 0.68 ppm for Ef and 0.25 ppm for Sf, 0.8 ppm for Fo, and 0.87 ppm for Hz. and were highly effective in field trials, comparable to standards indoxacarb and chloranthraniliprole, to control cotton thrips (Caliothrips indicus) nymphs than aldicarb at much lower rates. Furthermore, to evaluate their inhibitory action and cyclodiene cross-resistance, isoxazoline compounds were tested on both excitatory nicotinic acetylcholine receptors (nAChRs) and inhibitory GABA gated chloride channels and on Xenopus laevis oocytes expressing D.melanogaster RDL (resistance to dieldrin) GABA receptors. The results revealed that compound 80 potently blocked the GABA induced chloride currents in P.americana thoracic neurons with an IC₅₀ value of 10.8 nM and also produced dose-dependent inhibition of GABA receptors with IC₅₀ values of 75 and 95 nM for wild-type and mutant receptors (A302S) respectively (Fig. 24) [120].

Fig. (24).

4-heterocyclic aryl isoxazoline derivatives as GABA_A receptor modulators

(d). N & S-Heterocycles

(i). Thiazole derivatives

The potent anti-convulsant activity of certain 1,3-thiazolidin-4-one derivatives [121] inspired Pejovic et al. to design and synthesize derivatives of 1,3-thiazolidin-4-one by introducing ferrocene core as a bioactive pharmacophore. All the synthetics were evaluated for their anxiolytic properties in several in-vivo models like Light/Dark, Open field, Horizontal wire, and Diazepam-induced sleep tests. The results indicated that only compound 81 showed significant potency compared to known GABA targeting agents. Ligand docking experiment revealed that the high anxiolytic effect of compound 81 was attributed to its favorable interactions with the BDZ binding sites of the GABA_A receptor complex (Fig. 25) [122].

Fig. (25).

1,3-thiazolidin-4-one derivatives as GABA_A receptor modulators

Liu et al. synthesized a series of 4-substituted Thio-4-PIOL analogs to investigate the functional characteristics and inhibition of GABA-induced membrane potential in GABARs agricultural pest insect dieldrin receptors (RDLRs). Drosophila S2 cell lines cloned from small brown planthoppers (SBPs, Laodelphax straitellus F.) and common cutworms (CCs, Spodoptera litura F.) were used in the fluorescent imaging plate reader (FLIPR) membrane potential assays. Results showed that compounds 82 and 83 showed higher potencies as antagonists in CC RDLRs same as in SBPs RDLRs with IC₅₀ values 5.4 μM and 10.6 μM respectively. Their pharmacological properties were also studied in the GABARs of insect species, housefly (HF, Musca domestica L.), antagonism of HF RDL_ac GABA_ARs by synthesized molecules were examined by using Xenopus oocyte expression system. Inhibition of GABA-induced chloride current was determined by two Electrode Voltage Clamp technique. The results indicated that compounds containing 2-naphthyl ring (82) and 3-biphenylyl ring (83) reduced GABA-activated current (IC₅₀ 29.6 ± 1.7 μM and 20.4 ± 1.4 μM, respectively). To identify competitive antagonism, GABA concentration-response relationship of compound 82 was determined in the presence and absence of up to 30 μM of compound 82 and the results indicated that compound 82 showed decrease in potency of GABA in inducing currents at 30 μM. Furthermore, insecticidal activities of compounds 82 and 83 were determined by injecting them into adult female HFs and observations showed that both compounds showed low activity due to their high polarity. To investigate the interactions between 3-isothiazoles and insects GABARs, GABA, 82 and 83 were docked into the orthosteric site of HF RDLR homology model. Docking studies of both the compounds revealed the presence of an additional cavity on both sides of the core that connected Glu202 and Arg109 which could accommodate bulky aromatic substituents at 4-position of Thiol-4-PIOL. The 4-substituents formed hydrogen/π interaction with Tyr252 or Phe162. The two residues Glu202 (loop B) and Arg109 (loop D) interacted with the protonated nitrogen atom of piperidine ring and the deprotonated hydroxyl group of 3-isothiazoles respectively while the carbonyl oxygen atom of Ser203 showed hydrogen bonding interacton with the protonated nitrogen atom of piperidine ring (Fig. 26) [123].

Fig. (26).

4-substituted Thio-4-PIOL analogs as GABA_A receptor modulators

(e). Miscellaneous derivatives

In order to increase the GABA modulatory activity of the previously reported benz[e]indene and benz[f]indene compounds, [124–127] Scaglione et al. synthesized new cyclopenta[b]phenanthrene and cyclopenta[b]anthracene neurosteroid derivatives. The activity of these derivatives at GABA receptor was evaluated Electrophysiologically by using the in-vivo method and the binding ability of these derivatives was determined by the noncompetitive displacement of [³⁵S]-tert-butylbicyclophosphorothionate ([³⁵S]-TBPS) from the picrotoxin binding site on GABA_A receptor in rat brain membrane. Results showed that the cyclopenta[b]phenanthrene and cyclopenta[b]anthracene spiroepoxides, 84 and 85 showed potent displacement of ([³⁵S]-TBPS), comparable to benz[e]indene, benz[f]indene and neuroactive steroids with their IC₅₀ values 0.06 μM and 0.04 μM, respectively, whereas results of oocyte Electrophysiology stated that the compounds 84 and 85 potentiate the actions at α₁β₂γ_2L GABA_A receptor with their respective IC₅₀ values 1.53 ± 0.13 μM, 2.16 ± 0.09 μM, even at very low concentration (i.e. 0.1 μM) (Fig. 27) [128].

Fig. (27).

Cyclopenta[b]phenanthrene and cyclopenta[b]anthracene neurosteroid derivatives as GABA_A receptor modulators

It has been reported that neurosteroids that contain the 17β-acetyl group, as in allopregnanolone (86) and pregnanolone (87), are potent enhancers of GABA but their enantiomers are inactive [129]. Keeping this in view, Katona et al. synthesized new congeners comprising of the C-17 carbonyl group in place of the 17β-acetyl group [androsterone (88) and etiocholanolone (89), weak potentiators of GABA_A receptor function] [130]. Their enantiomers, as well as spiroepoxide compounds, were also synthesized by converting C-17 carbonyl group into the spiroepoxides. All the synthesized compounds were tested to check their binding ability towards GABA_A receptor by measuring the noncompetitive displacement of [³⁵S]-TBPS from a picrotoxin site on GABA_A receptors. The results of binding affinity revealed that the enantiomers of steroid 88 and 89 i.e. ent-88 androsterone and ent-89 etiocholanolone showed significant activity than their parent steroid with IC₅₀ values of 0.31 ± 0.04 μM and 0.38 ± 0.06 μM, respectively. Furthermore, the spiro-epoxides ent-90 and ent-91 displayed similar activity as steroid 86 and 87 consisting IC₅₀ values 0.47 ± 0.09 μM and 0.39 ± 0.04 μM, 0.074 ± 0.007 μM and 0.071 ± 0.018 μM respectively and also showed an increase in the Hill coefficient which indicated that there are additional binding sites on GABA_A receptors for these compounds. Moreover, oocyte Electrophysiology was also carried out, unfortunately, steroids showed less potency and efficacy than their enantiomers (Fig. 28) [131].

Fig. (28).

Androsterone and Etiocholanolone derivatives as GABA_A receptor modulators

Various structural attributes of previously reported 2β-aminopregnans derivatives acting on central nervous system [132] inspired Duran et al. to synthesize a new series of allopregnanolone (92) having a sulphamoyl moiety at C-2 position and either a hydroxyl group (compound 93 and 94) or fluoride at the 3α position (compound 95). The GABA_A receptor activity of these synthetic analogs was evaluated by assaying their effect on the binding of [³H] flunitrazepam, and [³H] muscimol and compared to the effect of neurosteroid allopregnanolone. The stimulation of [³H] flunitrazepam binding stated that the 3α-hydroxy-2,19-sulphamoyl analog 93 (EC₅₀ 63 ± 45 nM) and its N-benzyl derivatives (94) (EC₅₀ 81 ± 35 nM) were more potent than 92. It was noteworthy that the 3α-fluoro substituted analog 95 did not display any significant results for both binding assays performed. Furthermore, Ab-initio calculations stated that by the introduction of the sulfamidate bridge in the synthesized analogs, the relative alignments of the C-17 side chain and the 3α-hydroxyl were well maintained. Plausibly, the enhanced activity of the synthetic derivatives 95 and 94 was owed to the presence of sulfamide moiety which may enhance the hydrogen bonding interactions with the receptors. More interestingly, it was found that there was hydrophobic pocket present in the receptors which could accommodate bulky groups (e.g. benzyl ring of 94) (Fig. 29) [133].

Fig. (29).

Allopregnanolone derivatives as GABA_A receptor modulators

Allopregnanolone (92) and pregnanolone (96) are two endogenous neuroactive steroids with various pharmacological values (anticonvulsants, anxiolytics, and antistress). Their pharmacophore for positive modulation of GABA_A receptor includes COCH₃ or CN (hydrogen bond acceptor groups) on 17β position and hydroxyl group (hydrogen bond donor) at a 3α position which seems to be important for neuronal action. [134]. To examine this pharmacophoric effect, Covey and coworkers previously examined nonsteroidal analogues (perhydro benz[e]Indenes) of 92 and 96 that mimicked parts of the steroid nucleus and stated that the replacement of ring-A having 3-hydroxy group with an open chain of appropriate length could mimic steroids having either a 3α or a 3β hydroxyl, and being able to bind to the potentiating and the inhibitory sites on the GABA_A receptor [135]. Inspired by these findings, M.V. Dansey et al. synthesized a series of A-homo steroids related to 92 and 96 by incorporating seven-membered rings (Ring A). Compounds were evaluated for their inhibitory effect by binding [³⁵S]-tert-butyl-bicyclo[2.2.2]phosphorothionate [TBPS] assay using allopregnanolone as a positive control. Interesting results were obtained by the assay which revealed that alpha-oriented hydroxyl group on ring-A exhibited 50–75 percent maximal inhibition of TBPS with IC₅₀ values in the micromolar range. From the series of synthesized compounds, 97–99 showed interesting results amongst which 98 displayed an IC₅₀ similar to that of allopregnanolone however its maximal inhibition was lower. The study emphasized the importance of the double bond present in 5^th and 6^th carbon for activity as its reduction led to a 10-fold decrease in activity as seen for compound 100 (Fig. 30) [136].

Fig. (30).

Allopregnanolone and Pregnanolone steroidal derivatives as GABA_A receptor modulators

Previously, Hogenkamp et al. developed a series of novel and efficient enaminone amide GABA receptor modulators (101) and reported that the compounds interact with novel sites on GABA_A receptors [137]. To rationalize the detailed description of these binding sites, Jin Cheng et al. studied the binding interactions between enaminone amides with novel sites of GABA_A receptors by using in-vitro computational studies. Homology model of rat α1β2γ2 GABA receptor was obtained using cryo-electron microscopy structure of nicotinic acetylcholine receptors as a template. Molecular docking and pharmacophore analysis was carried out to identify the critical residues involving the recognition of the ligands. The results of molecular docking study indicated that His 128, Tyr 186 and Tyr 236 of α subunit were essential to form H-bond interaction with the 4-chlorophenylamino, acrylamide, and 2-chlorobenzoyl group of ligands. (Fig. 31) [138].

Fig. (31).

Enaminone amides as GABA_A receptor modulators

Keeping in view the vast biological applications of Honokiol (102) (a neolignan compound isolated from Magnolia officinalis), Taferner et al. carried out its structural modification of and synthesized new derivatives to determine their effect on seven GABA_A receptors composed of different subunits in Xenopus laevis oocytes and analyzed the modulation of chloride current by using two-microelectrode voltage clamp techniques. Honokiol was found to be most efficient for α₃ β_2, which showed maximal (I_GABA) enhancement (2386%) > α₂ β₂ (1130%) > α₁ β₂ (1034%) > α₁ β₁ (260%) with its EC₅₀ values ranging from 23.4 μM (α₅ β₂) to 59.6 μM (α₁ β₃). Compound 103 remarkably enhanced chloride current (I_GABA) by 26.01%, as well as showed higher subtype selectivity towards α₁ β₂ receptors with an EC₅₀ value of 3.8μM which revealed that acetamido group at R⁵ played an important role to activate the receptor. A well-established structure-activity relationship obtained from the assay is given in (Fig. 32) [139].

Fig. (32).

Honokiol derivatives as GABA_A receptor modulators

To study the inverted binding orientations of androsterone enantiomers (104) Krishnan et al. synthesized steroids and their enantiomers and the binding orientation of the enantiomers at GABA_A receptors sites were deduced using enantiomers containing OBn substituents at either C-7 or C-11 positions. All the synthetics were evaluated for their ability to displace [³⁵S]-tert-butylbicyclophosphorothionate ([³⁵S]TBPS) from the picrotoxin binding sites on the heterogeneous GABA_A receptors found in rat brain membrane [140]. Furthermore site-directed mutagenesis study [140] was carried out to examine the effect of Q241L mutation in the α1 subunit on the action of 5β-reduced steroid potentiator and its active enantiomers. The binding of enantiomers at the same receptor sites as well as their ability to potentiate chloride current mediated by GABA at α₁β₂γ_2L GABA_A receptors in X. laevis oocytes was also evaluated. Binding results showed that the steroids 104, 105 and ent-106 were more potent displacers with their IC₅₀ values 15 ± 2 nM, 52 ± 5 nM and 74 ± 7 nM respectively (Fig. 33) [142].

Fig. (33).

Steroidal analogues as GABA_A receptor modulators

Rueda et al. previously identified three structurally related stilbenoids (compounds 108–110) from Pholidota chinensis (Orchidaceae) [143–145]. Compounds were tested for their GABA_A modulatory activity in the Xenopus laevis oocytes assay at GABA_A receptors of the subtype α₁β₂γ_2s. Among the three compounds, only compound 109 (Batatasin) displayed strong GABA_A receptor modulatory activity with high efficiency (1512.9 ± 176.5%) and potency (52.5 ± 17.0 μM) and was shown to be a allosteric modulator at α₁β₂γ_2s receptors with higher efficiency than Benzodiazepines. Conformational flexibility was censorious for modulatory activity of stilbenoids. To confirm the influence of double bond, other stilbenoids, and their corresponding dihydro derivatives were tested in the Xenopus laevis oocytes assay which showed that dihydrostilbenes (110 and 111) showed maximal potentiation of I_GABA 870.7 ± 106.8% and 694.2 ± 86.0% and potency with their EC₅₀ values 54.5 ± 13.4 μM and 20.2 ± 6.4 μM due to the replacement of hydroxy group at C-3 and C-5 with bulkier oxygenated functions. Biological data revealed an interesting structure-activity relationship describe in figure 34 [146].

Fig. (34).

Stilbenoids derivatives as GABA_A receptor modulators

To enhance the modulatory potential of piperine derivatives (112) Schoffmann et al. carried out the structural modification at the amide nitrogen and the diene of the lead compound to synthesize a series of new piperine analogs which were evaluated for their effects on GABA_A R expressed in Xenopus laevis oocytes by means of two-microelectrode voltage-clamp technique. Modification at the linker part of piperine did not affect GABA_A receptors. Among all the synthetics compounds, 113 (most efficient), 114 (most potent) and 115 (previously reported compound) [146] induced the strongest modulation of I_GABA at α₁β₂γ_2s GABA_A than piperine (113; I_GABA-max : 1581 ± 74 %, EC₅₀ = 51.7 ± 9.5 μM, 114; 1673 ± 146% and EC₅₀ = 13.8 ± 1.8 μM, 115; 378 ± 15 %, EC₅₀= 21.5 ± 1.7 μM and 112; 302 ± 27 %, EC₅₀= 52.4 ± 9.4 μM). This indicated that by replacing the cyclic piperidine residue with noncyclic substituents containing 3–4 carbon atoms at tertiary amide, improved both efficiency and potency of the compounds such as N,N-dipropyl (113), N,N- dibutyl (114) and N,N-diisobutyl (115). Furthermore, to abolish the activation of TRPV1 (because it causes some side effects such as changes in pain sensation, body temperature and induction of fear that interfere with GABA_A- mediated effects), compounds 113 and 114 were studied for their interaction with TRPV1 expressed in X.laevis oocytes. Compound 113 displayed strong anxiolytic effects on mice treated at doses ≥ 0.3mg/kg body weight for their ability to spend more time in the open arm (OA) of the elevated plus maze test compared to the saline-treated control group (control, 28.7 % ± 2.7 %, n=41; 113, 45.6 % ± 3.2 %, n=17, p< 0.001 ) (Fig. 35) [148].

Fig. (35).

Piperine analogs as GABA_A receptor modulators

To enhance the potency as well as the efficacy of Magnolol and 4’-O-methylhonokiol [149], Fuchs et al. synthesized analogs of these two natural products as potential allosteric potentiators (PAM) of GABA_A receptors. The ability of the synthesized compounds to potentiate the GABA-induced current was determined at recombinant α₁β₂γ_2s GABA_A receptors in Xenopus oocytes. Results revealed that compounds 116–119 were most potent PAM for potentiation of GABA-induced currents in the range of 1000 %, 5000 %, 4000 % and 3000 % at 10 μM and also identified as allosteric modulators. Furthermore compounds 116 and 119 were tested at different subtype GABA_A receptors and also at receptors containing point mutations (α₁β₂N265Sγ₂, α₁β₂V436Tγ₂) to identify whether newly synthesized compounds shared same binding sites. The results stated that the current potentiation was largely influenced by changing β₂ with β₁ than α subunit in both compounds 117 and 118, based on which it was cleared that both the compounds (117 and 118) did not share their binding sites with other potentiators. Summary of this SAR study is shown in Figure 36 [150].

Fig. (36).

Magnolol and 4’-O-methylhonokiol analogs as GABA_A receptor modulators

Eltahawy et al. isolated new ceramides (120) from Red sea soft corals of Sacrophyton auritum which were tested for their anxiolytic activity using Light-Dark transition box and Elevated Plus Maze as well as for their in-vivo anticonvulsant activity by using pentylenetetrazole (PTZ)-induced seizure model. Furthermore, molecular modeling studies were also carried out to identify the mechanism of the compounds. Results of docking study indicated that compound 120 showed binding interaction with the GABA receptors similar to Benzodiazepines (Flurazepam). The secondary hydroxyl group of compound 120 showed hydrogen bonding with Thr102. It was observed that compound 120 accommodated at the surface of GABA allosteric sites without any steric collision whereas docking of compound 120 in 5-HT₃ receptors showed that compound 119 docked well in the active site and formed hydrophobic interactions with W53, W145, Y193, Y186, D162. Docking with 5-HT₃ receptors showed that compound 120 acts as a potent but not a selective blocker of 5-HT₃ receptor. These comparative docking studies indicated that compound 120 was a GABA receptor selective inhibitor as compared to serotonin receptors. (Fig. 37) [151].

Fig. (37).

Miscellaneous derivatives as GABA_A receptor modulators

Inspired from the previously reported work on 3-acetylamino-4’-O-methylhonokiol (AMH, 121), honokiol (H, 122) and 4’-O-methylhonokiol (MH, 123) [152], Bernaskova et al. synthesized a series of nitrogenated honokiol derivatives with the aim to elucidate structure-activity relationships by analyzing allosteric modulation and agonistic effects on α₁β₂γ_2s GABA_A receptors. In previous papers, it has been found that AMH showed maximal potentiation of I_GABA through α₁β₂ receptors. All the synthetics were evaluated for their concentration-dependent enhancement of I_GABA and compounds 124 and 124 showed strongest I_GABA enhancement at GABA_A receptors with Emax (123.4 ± 9.4% and 117.7 ± 13.5%). Compound 124 also displayed higher potency to induce chloride current through α₁β₂γ_2s GABA_A receptors. Moreover, honokiol and its nitrogenated derivatives 121, 122, 123 and 126 induced significant inward chloride current even in the absence of GABA. AMH (121) Honokiol (122), and compound 124 were strong partial agonist compared to compound 125 with their I_GABA-max 63 ± 6%, 78 ± 6%, 59 ± 1% and 52 ± 1% respectively. While compounds 123 and 126 were weakest partial agonists, they were still more potent than full agonist GABA with EC₅₀ values 6.9 ± 1.0 μM and 33.2 ± 5.1 μM (Fig. 37) [153].

By considering the various biological facts of natural products magnolol (127) and honokiol (128) (Magnolia officinalis) [154], Rycek et al. synthesized new unsymmetrical derivatives of magnolol and honokiol via metal-assisted cross-coupling reaction. In Magnolol and honokiol structure (biaryl isomeric molecule), aromatic ring A is identical while ring B differs in the position of the hydroxyl group. In this paper structural modifications were carried out only at ring B. Previously unsymmetrical derivatives were obtained via bromination of anisole and then Suzuki coupling with phenylboronic acid further modification was carried out to obtain derived compound (4-allyl-2-bromoanisole) which was used as intermediate for the synthesis. However, the major drawback of this synthetic route was the over bromination of double bond. This limitation was overcome by a two-step reaction which mainly relies on palladium-catalyzed transformation followed by Suzuki coupling. All the synthetics were tested for their ability to modulate α₁β₂γ_2s GABA_A receptors at low concentration of GABA in Xenopus oocytes. Compounds 129 and 130 containing p-methoxy group and m-methoxy carbonyl group showed higher efficacies than natural product honokiol at 3 μM with their %I_GABA-max values 162 ± 31 % and 443 ± 60 % respectively. Furthermore, the effect of 129 was tested in a mutant for α₁β₂N265Sγ_2s receptor and it was observed that there was a significant decrease in response which ensured that β₂N265 play an important role in β-subtype selectivity. The SAR study is summarized in Fig. 38 [155].

Fig. (38).

Magnolol and honokiol analogs as GABA_A receptor modulators

2.1.2. GABA_B receptor modulators

To study the function and localization of GABA_B receptors on living cells Li et al. designed and synthesized trimodular activity-based probes incorporating three basic moieties in their chemical architecture i.e. the photoinduced cross-linking group (trifluoromethylaryldiazirine)[156], a fluorophore (4,4–difluoro-4-bora-3a,4a-diaza-s-indacene) [157] and GABA_B receptors binding group containing diazirine. Design of 131 was based on structure 132, a GB1 selective high-affinity antagonist. [28,158]. Furthermore, 133 was also synthesized which was composed of only the photoaffinity and fluorophoric groups. Since compound 133 did not contain any bioactive ligand, it failed to label the GBI expressed on CHO cell surface. Before performing labeling studies, these probes were tested for their bioactivity as GABA_B receptor antagonists where 131 significantly retain its affinity towards GABA_B receptors with an IC₅₀ value of 1.03 μM. 131 was further evaluated for its capacity to label the GABA_B receptors. In this assay Chinese hamster ovary (CHO) cells were employed. This probe exhibited strong binding affinity at the ligand binding pocket of GB1 subunits and also labeled the GB1 subunits of GABA_B receptors on the cell surface in an activity-based manner. Furthermore, the importance of covalent bond formed between 131 and GABA_B receptors induced by UV irradiations revealed that the labeling efficacy of the probe was increased even at a low affinity for its target protein (Fig. 39) [159].

Fig. (39).

Trifluoromethylaryldiazirine based derivatives as GABA_B receptor modulators

To improve the potency of R-(−) enantiomers of Baclofen (134) [160–164], Xu et al. synthesized a series of R-baclofen analogs by introducing various groups at the 3^rd position of the phenyl ring and investigated them to evaluate in-vitro and in-vivo potency towards GABA_B receptors. Firstly, compounds were assessed for their in-vitro potency as agonists at GABA_B receptors present in HEK cells. The activation of GABA_B receptors mainly depends upon the increased level of cytoplasmic Ca²⁺ released from intracellular stores which are detected by the Ca²⁺ sensitive fluorescent probe and expressed in pEC₅₀ values. The GABA_B agonistic activity of the most potent compound was further characterized by determining its ability to induce GABA_B receptor mediated G-protein activated inwardly rectifying (GIRK) K⁺ current which showed that compound 135 stimulated GIRK current at very low concentration than the parent molecule. The in-vivo pharmacology of GABA_B agonistic activity was also determined using mouse hypothermia model [165]. The results of both in-vitro and in-vivo assay showed that compound 135 containing 4-pyridyl ring was more potent than the R-baclofen with pEC₅₀ values 8.5 and 7.1 respectively. SAR studies of this group of compounds is summarized in Fig. 40 [166 ]. Furthermore, to overcome the major limitation of baclofenac i.e, short duration of action, rapid tolerance and narrow therapeutic window (muscle relaxation, sedation), poor blood–brain barrier penetration (Bowery, 2006), Malherbe et al. characterized the derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as potent allosteric enhancers of GABAB receptors by determining the enhancing potency using Fluorometric Imaging Plate Reader (FLIPR) assay and GTPᵞ[³⁵S] binding assay. The results of both assays indicated that the compound 136 (+) enantiomers of rac-BHFF showed a highest enhancing effect of GABA as well as baclofenac activities at native and recombinant GABAB receptors in rat hippocampal slices than (−) enantiomers. After the carefully examination of in vitro assays, the in vivo testing of antianxiety potential of rac-BHFF were also carried out in mice. The results of in vivo mechanism based model revealed that rac-BHFF of compound 136 showed anxiolytic activity in the stress induced hyperthermia (SIH) model in mice.

Fig. (40).

R-baclofen analogs as GABA_B receptor modulators

2.1.3. GABA_C receptor modulators

It has been shown that selective antagonism of GABA_C receptors enhances cognition and memory [40]. 1,2,5,6-tetrahydropyridine-4-yl-methyl-phosphinic acid (TPMPA) (137) was the first reported GABA_C receptor selective antagonist [167]. The design of this potent antagonist was based on the structural features of isoguvacine (138) and 3-aminopropyl(methyl)phosphinic acid (139) which are active at GABA_A-GABA_C and GABA_B-GABA_C receptors, respectively. The structure of TPMPA was also attributed to as a bioisostere of potent but non-selective GABA_C receptor antagonist 140. To obtain selective GABA_C receptors antagonists, a series of cyclopentene and cyclobutane phosphinic acids were synthesized, taking TPMPA (139) as a lead compound and their potency and selectivity within the GABA receptors subtypes was investigated. The SAR study revealed that the efficiency of the synthesized compounds generally depends upon phosphinic acid substitution, that exhibited activity in the decreasing order of methyl > ethyl > n-butyl > isopropyl > benzyl. The effect of stereochemistry at 4^th position of these compounds was also investigated by determining their dose-response curve. Among all the synthetics, compound 141 exhibited potent antagonistic activity at ρ_1-GABA_C receptor only in the form of (S)-enantiomer (Fig. 41) [168].

Fig. (41).

Cyclopentene and cyclobutane phosphinic acid derivatives as GABA_C receptor modulators

2.2. GABA transporter (GAT) or GABA uptake inhibitors

It has been reported that pyrrolidine derivatives containing (2S)-homoproline moiety exhibited prominent GAT-1 (GABA-uptake proteins) inhibitory activity.[169] Based on these findings, Zhuang et al. followed their previously reported work to synthesize (2S)-4-fluoroproline and (2S)-4-fluoropyrrolidine-2-acetic acid enantiomeric derivatives by replacing hydroxyl group at position C-4 with fluorine atom due to its distinctive properties such as high electronegativity, strong lipophilicity, extremely low polarizability and hydrogen bond forming potential as found in preliminary studies [170–174]. Synthesized derivatives were evaluated for their inhibitory potential against GAT-1 proteins on culture cell lines expressing mouse GAT-1 in transport bioassay by using tiagabine as a control. In vitro results revealed that fluorinated compounds showed a slight improvement in the inhibitory potency as compared to their 4-hydroxy compounds (142 vs 143 and 144 vs 145) but they were still weaker compared to (GAT-1 IC₅₀ = 0.159 μM). The assay also figured out an interesting fact that C-4 fluorine compounds did not influence the affinity towards the GAT-1 proteins, most probably due to the reduction in basicity of the compounds owed to strong inductive effect from fluorine atom (Fig. 42) [175].

Fig. (42).

Proline and pyrrolidine-2-acetic acid derivatives as GAT inhibitors

Inspired by previously reported pyrrolidine derivatives that showed potent GABA uptake inhibitory activity (146 and 147) [169,170], Zhao et al. synthesized a new series of pyrrolidine derivatives in a stereoselective pure forms. These synthesized derivatives were evaluated for their inhibitory potency against GAT1 and GAT3 GABA transporter protein. The compounds were tested by using subcellular membrane fractions from frontal cortex (bfcP2B) and brain stem (bbsP2C) of bovine brain. Results obtained from the assay indicated that the compounds devoid of lipophilic N-substituents lacked inhibitory potency even at higher concentrations (up to 100 μM). Among all the synthetics compounds (2S,4R)-148 (GAT3: IC₅₀ = 29.7 μM, GAT1: IC₅₀ = 45.1 μM) and (2S,4R)-149 (GAT3: IC₅₀ = 66.7 ± 11.2 μM, GAT1: IC₅₀ = 48.4 ± 1.6 μM) showed potent inhibitory activity against GAT1 and GAT3 (Fig. 43) [176].

Fig. (43).

Pyrrolidine derivatives as GAT inhibitors

In continuation from previous work with pyrrolidin-2-yl-acetic acid acting as strong GAT (GABA transporters) inhibitors, Steffan et al. synthesized a series of racemic (rac) 2-substituted pyrrolidine-2-yl-acetic acid based potential inhibitor (RS)-150 [171] and N-arylalkyl derivatives by substituting alkyl, hydroxyl, and amino group at 2-position of pyrrolidine derivatives. The congeners were examined for their inhibitory potency as well as subtype selectivity at mouse GAT1 to GAT4 GABA transporter protein by using [³H] GABA uptake assay. The assay revealed some interesting aspects of SAR which are summarized in Fig. 44 [177].

Fig. (44).

2-substituted pyrrolidine-2-yl-acetic acid derivatives as GAT inhibitors

It has previously been reported that nipecotic acid (153; GABA uptake inhibitor) lacks the capability to cross the blood-brain barrier (BBB) due to its low lipophilicity [178]. A huge number of compounds have been reported (154–156) so far by introducing lipophilic side chain on the nitrogen atom of nipecotic acid with improved BBB penetrability as well as GAT1,3 selectivity [179–183]. It has been found that, the presence of 4,4-diphenylbut-3-enyl and 4,4-bis(3-methylthiophene-1-yl)but-3-enyl as in 154 and 155 (tiagabine) are crucial for GAT-1 inhibition while 2-[tris(4-methoxyphenyl)methoxy]ethyl residue is responsible for GAT-3 inhibition. Similarly, 3-aminopropanoic acid (157) and methyl substituted 3-aminopropanoic acid (158) are GABA uptake inhibitors in glial cells and exhibit reasonable potency against GAT-1 and GAT-3 subtypes [184,185]. To improve their selectivity and permeability, Sitka et al. synthesized a series of N-monoalkylated derivatives of 157 and 158 by introducing large lipophilic side chains in a similar manner as in compounds 156-158. All the synthesized compounds were tested for their inhibitory potency at against GAT-1 and GAT-3 using HEK293 cell lines and exhibited inhibitory potential similar to the parent lead molecules 157 and 158 (Fig. 45) [186].

Fig. (45).

3-aminopropanoic acid derivatives as GAT inhibitors

For the development of mGAT3 selective GABA uptake inhibitors, Hack et al. synthesized three new series ω-imidazole-2-yl, 4-yl and 1-yl alkanoic and alkenoic acid based on the structure of 1H-imidazol-4-ylacetic acid by modifying the length and position of the side chain. Synthetics were evaluated in [³H] GABA uptake assays for their inhibitory potency for four subtypes of GABA transporters viz. mGAT1–4 expressed in HEK cells. The results indicated that most of the synthesized compounds inhibited GABA uptake with high potency and interestingly the compounds 159–162 showed higher affinity as well as good subtype selectivity to mGAT3 transporter with pIC₅₀ values of 4.54 ± 0.15, 4.76 ± 0.08, 4.64 ± 0.15, 4.59 ± 0.02 respectively. (Fig. 46) [187].

Fig. (46).

Imidazole alkanoic and alkenoic acid derivatives as GAT inhibitors

Two series of new structures (series A and B) were designed and synthesized by Kulig et al. using 4-hydroxybutyric acid 163 (GHB) (a neurotransmitter inhibitor) as a core structure [188,189], in which carboxylic acid functional group was modified to more lipophilic groups and N-diphenylmethylpiperazine was introduced at 2^nd position. All the synthetics were tested at four murine GABA transporters subtypes GAT1-GAT4 using [³H] GABA uptake assay. The affinity of GAT1 was determined by MS-binding assay with NO711 as a non labeled marker [190]. The biological evaluation revealed that all the compounds were active amongst which compound 164 from the series A, exhibited potent inhibition against GAT3 with IC₅₀ value 9 μM, while 165 and 166 showed moderate potency against GAT1 with IC₅₀ values of 22 and 14 μM, respectively. The comparison between two series revealed that the introduction of fluorine atom on N-diphenylmethylpiperazine leads to decrease in activity profile while the benzyl substituents in the amide group and benzhydralpiperazinyl are crucial for the activity (Fig. 47) [191].

Fig. (47).

N-diphenylmethylpiperazine derivatives as GAT inhibitors

Kowalczyk et al. synthesized a series of new 4-aminobutanamide derivatives and evaluated for their in vitro inhibitory activity and selectivity towards cloned murine GABA transporter mGAT1- mGAT4 in uptake assay. The mGAT1 affinity was also evaluated by using MS binding assay. It was shown previously that 2-substituted-4-hydroxybutamide containing phthalimide group at 4-position showed anticonvulsant activity [192] as well as antinociceptive properties [192)] while 4-(1,3-dioxoisoindolin-2-yl) butanamide with 2-(4-benzhydryl)-piperazin-1-yl residue showed less GAT inhibition[192]. Hence, new derivatives were obtained with primary amine group at 4-position. In vitro inhibitory activity was carried out by [³H] GABA uptake in HEK-293 cells to identify GABA uptake inhibitors and the results showed that the compounds 167, 168 and 169 showed highest potency towards mGAT2 with their pIC₅₀ values 5.16 μM, 5.16 μM, 5.23 μM respectively. Furthermore, 170 was selected for pilocarpine-induced seizure test, forced swim test and hot plate test. The results showed that compound 170 displayed the most potent activity against mGAT4 with significant anticonvulsant activity and decreased locomotor activity at a dose of 30 mg/kg with significant antinociceptive activity. SAR studies indicated that introduction of benzyl substituents on the amide group and aromatic and lipophilic substituent at 2-position of 4-hydroxybutanamide are pivotal for the activity and the presence of tertiary amino group is necessary for their effective interaction with GABA uptake system (Fig. 48) [193].

Fig. (48).

4-Aminobutanamide derivatives as GAT inhibitors

2.3. GABAergic enzyme inhibitors

In continuation from the previous work [194], Silverman et al. synthesized a conformationally rigid analogue of vigabatrin (171), a mechanism based inactivator that avoids the Michael addition inactivation pathway (a pathway responsible for the formation of byproduct which acts as a reactive electrophile responsible for retinal toxicity, a major drawback of vigabatrin). This mechanism-based inactivator structurally related to the vigabatrin act as an unreactive compound which converts the inactivator into the species that leads to inactivation of the GABA-amino transferase (GABA-AT) prior to escaping of that species from the active sites. All the synthetics were tested to measure the dopamine release by in vivo micro positron emission tomography (micro-PET) and their effect on the release of dopamine in the nucleus accumbens (NAcc) in freely moving rats was determined. Among all the synthetics, compound 172 (CPP-115, Ki 9.7 μM) was found to inactivate GABA-AT instantaneously than vigabatrin (Ki 850 μM, even in the presence of electron withdrawing group (β-mercaptoethanol). Furthermore, compound 172 was potent in in vivo in a rat model for drug addiction as well as in the treatment of infantile spasms in the rat model (Fig. 49) [195].

Fig. (49).

Vigabatrin analogs as GABA-AT inhibitors

In continuation from the previous work strictly related to cyclopropane compounds that bind selectively to structure-unknown targets proteins with stereochemical diversity-oriented conformational restricted strategy [196], Nakada et al. synthesized a series of cyclopropane based conformationally restricted GABA analogs. Because of its low molecular weight and hydrophilic structural features suitable for an efficient lead, trans and cis-2,3-methano analogs, trans and cis-3,4-methano analogs and their enantiomers were used to develop new series of compounds. All the synthesized compounds were tested for their inhibitory potency on GABA uptake in GAT1/CHO, GAT2/CHO, GAT3/CHO cells and betaine GABA transporters (BGT1/CHO) cells. Among all the synthetics, only analog 173 showed potent inhibitory effects on GAT3 and BGT1 with IC₅₀ values 92.6 ± 2.2 μM and 92.8 ± 0.7 μM, respectively. Furthermore, the binding affinity of synthesized compounds to GABA_B receptors with crude synaptic rat brain in the presence of isoguvacine was also rationalized. Anticonvulsive effect of the GAT1 inhibitor, 173 were also examined. The results of anticonvulsant effects showed that compound 173 effectively prolonged the latency of clonic convulsion (ED₅₀ = 189 ± 104 nM) in the mouse model whereas binding assay revealed that analog 173 showed high affinity for GABA_A receptors with Ki values 0.34 μM (Fig. 50) [197].

Fig. (50).

Cyclopropane based conformational restricted GABA analogs as GABA-AT inhibitors

Keeping in view the potent activity against GABA-amino transferase (GABA-AT) of 5-fluoro-4-aminopentanoic acid (174) [198] and vigabatrin (171) (hexanoic acid skeleton), [3,199,200] Juncosa Jr. et al. synthesized three analogues of 174 and tested for time-dependent inhibition of GABA-AT by using Vigabatrin as a positive control. Unexpectedly, these synthesized compounds were unable to inactivate or inhibit GABA-AT. Molecular modeling studies were carried out to speculate the reason behind the inactivation of synthesized compounds by using the previously published X-ray crystal structure of GABA-AT inactivated by Vigabatrin [201]. Docking studies of synthesized compounds 174, (S,S)-175, 176 and 171 indicated that there was existence of an accessory-binding pocket near to the main active site of enzyme, that occupied the vinyl group of vigabatrin but it was too narrow to acquire the extra width of the distal methyl group of the synthesized compounds because of the alternative conformation, thus giving rise to unfavorable steric interactions with the proteins (Fig. 51) [202].

Fig. (51).

5-fluoro-4-aminopentanoic acid derivatives as GABA-AT inhibitors

Keeping in view the biological aspects of both benzofuran and amide Shakya et. al. designed and synthesized new series of compounds containing benzofuran moiety attached with the various amines with greater binding interaction with GABA-AT. It was also reported that a good anticonvulsant compounds must have hydrophobic aryl ring, a hydrogen bonding domain, an electron donor acceptor system and another hydrophobic aryl ring, mainly responsible for the metabolism as well as showed higher binding affinity [203–205]. All the synthetics were tested for their anticonvulsant potential using phenytoin as a reference drug and their ED₅₀ were also calculated. The results of anticonvulsant activity indicated that compound 177exhibited the higher anticonvulsant potential against maximal electroshock induced seizures (MES) as compared to the phenytoin with ED₅₀ values 0.72 and 1.00 mmol kg⁻¹ respectively. Furthermore, to understand their mechanism of action, the GABA level in brain was determined, clobazam was used a reference drug and the results revealed that the compounds 177 significant increased the level of GABA in brain as compared to the reference drug. To understand the binding interaction of the most active compound 177 with the GABA-AT enzyme binding site, a molecular modelling study was carried out and results indicated that compound 177 showed high binding affinity at GABA-AT by showing interaction with the Arg192, Ser137, Thr353 and Lys329 amino acid residue of GABA-AT enzyme (Fig. 52) [206].

Fig. (52).

Benzofuran-acetamide derivatives as GABA-AT inhibitors

3. Completed Clinical trials of GABA modulators, transporters, and metabolic enzyme inhibitors.

The successful validation of a number of GABA modulators in neurodegenerative disorders has catalyzed the development of selective molecules which are undergoing clinical trials. Table 1 focuses on advances made completed Clinical trials of GABA receptor modulators [208].

Table 1.

Various GABA receptor modulators, uptake inhibitors and metabolic enzyme inhibitors that have completed clinical trials [208]

S. NO.	COMPOUND	STRUCTURE	CONDITION	COMPANY	ADMINISTRATION ROUTE	STARTING DATE	PHASE	STATUS
1	AZD7325 AZD-7325 + Flumazenil Flumazenil		Healthy Healthy Anxiety Exposed to Anesthesia Hypersomnia, Primary Hypersomnia, Idiopathic Hypersomnia, Narcolepsy without Cataplexy Tourette Syndrome	University College, London University College, London Astrazeneca, Sweden University of Sao Paulo Lynn Marie Trotti, Georgia Research Alliance National Institute of Neurological Disorder and Stroke (NINDS)	Oral Oral Oral or IV IV Oral IV	Feb-2016 Sep-2014 Feb-2008 Jan-2011 Sep-2010 Apr-2002	PHASE-I PHASE-I PHASE-I Unknown PHASE-I, II Unknown	Completed on July-2016 Completed on Aug-2015 Completed On July-2008 Completed on Sep-2012 Completed on Jan-2012 Completed on Mar-2010
2	Methylphenidate		Cognition Disorder, Attention Deficit Disorder, Traumatic Brain Injury	National Institutes Of Neurological Disorders And Stroke (NINDS), United States	Oral	Aug-2014	PHASE-II	Suspended on Feb-2016
3	Midazolam		Child Anesthesia Morbidity Delirium on Emergence	Korea University Anam Hospital	IV	Aug-2012	Unknow n	Completed on Jan-2013
4	PF-06372865		Healthy	Pfizer, United States	Oral	Apr-2014	PHASE-I	Completed on Sep-2014
5	Baclofen		Brain GABA-B Function Alcohol Dependence Autism CHEYNE Strokes Respiration	Imperial College London, Medical Research Council, London, United Kingdom Charite University, Berlin, Germany Gonzalez-Heydrich, Joseph, M.D. Boston Children’s Hospital Assistance Publique-Hopitaux de Paris, France	Oral Oral Oral Oral	July-2013 Feb-2011 Oct-2010 Mar-2010	Unknown PHASE-II PHASE-I PHASE-I	Completed on Sep-2014 Completed on May-2014 Completed on July-2012 Completed on Sep-2011
6	Dexmedetomidine		Inhalational Anesthetics Adverse Reaction, Delirium on Emergence, Strabismus Following Ocular Surgery	Yao Yusheng, West China Hospital, Fujjan, China	Intranasal	Sep-2013	PHASE-IV	Completed on Aug-2014
7	Etomidate + Meperidine		Choledocholithiasis Cholangiocarcinoma Pancreatitis Pancreatic Cancer	Cheju Halla General Hospital, Korea	IV	Apr-2013	PHASE-IV	Completed on Aug-2013
8	Clobazam		Electrical Status Epilepticus in Sleep	Boston Children’s Hospital, Boston, Massachusetts, United States	Oral	July-2012	Unknown	Completed on July-2015
9	Clarithromycin		Hypersomnia, Idiopathic Hypersomnia, Narcolepsy	Lynn Marie Trotti, Emory Sleep Centre, Atlanta, Georgia, United States	Oral	July-2010	PHASE-II	Completed on Sep-2012
10	Clonazepam		Neuropathic Pain	University Hospital, Geneva, Switzerland	Oral	Apr-2010	PHASE-I	Completed on Nov-2011
11	Tolterodine		Neuropathic Pain	University Hospital, Geneva, Switzerland	Oral	Apr-2010	PHASE-II	Completed on Nov-2011
12	Gabapentin		Pruritus	Prince of Songkla University, Hat Yai, Songkhla, Thailan	Oral	Sep-2009	Unknown	Completed on Nov-2010

4. Conclusion

In view of a significant number of reports on GABA receptor modulation, transport, and metabolic enzyme inhibition, this review article presents the rational approaches behind the design of molecules exerting modulatory effects on GABAergic activity. The article also highlights the SAR along with mechanistic insights revealed during the investigation of these analogs. In some of the cases, molecular modeling studies have played a key role in identifying the interactions responsible for the binding of designed molecules with the amino acid residues of GABA receptors and GATs. Numerous molecules have displayed significant results and their promising potential clearly places them ahead as potential future drug candidates. Particularly, among the above-mentioned GABA_A receptor modulators, the molecular structure of compound 24 is quite stable to oxidative cleavage, resulting in excellent in-vitro metabolic stability in biological conditions. The high functional efficacy and GABA_A binding selectivity of compound 24, offers a prominent starting point for lead optimization to further develop novel drug compounds for the treatment of insomnia.

The second hit lead molecule found among the GABA_A modulators is AZD7325 (48) which currently is in Phase II clinical trials for the treatment of anxiety. It is found safer to use than lorazepam as its 2 mg dose is sufficient to occupy 50 percent of GABA_A receptors. Therefore, it has mitigated side effect pattern, with potentially lower neurophysiological and cognitive side effects in comparison to non-selective Benzodiazepines [207].

3-(4-pyridyl)methyl ether (134) that has been shown to be almost 50-fold potent than R-baclofen at rodent and human GABA_B receptors when tested in vitro. Mouse hypothermia studies confirmed that 134 crosses the blood-brain barrier easily and is approximately 50-fold more potent in vivo than baclofen after systemic administration, which could be used to treat CNS disease conditions.

Amongst the fewest reports on GABA uptake inhibitors, compound 168 displayed significant antinociceptive activity in the hot-plate and also decreased the locomotor activity in animal model at a dose of 30 mg/kg. In account to the sedative action of 168, the prolongation of the latency time to pain reaction is likely to be a false positive effect resulting from sedation and reduced the number of seizure episodes by 83% at the dose level of 30 mg/kg.

Besides above information, the compound 170 (CPP115) showed excellent in vivo activity in rat model for drug addiction and also showed 100 times more effectiveness in the treatment of infantile spasms in a rat model than vigabatrin. In past, Vigabatrin is primarily used for the treatment of seizure disorder but this drug showed a serious adverse side effects that mainly associated with the long term vigabatrin treatment. Recently, it was also reported that CPP-115 considered as a novel GABA-AT inhibitor with higher potency, specificity, tolerability and ocular safety than vigabatrin and also showed same mechanism of action as vigabatrin. CPP-115 is considered as a hit lead molecule for further investigations and is licensed to Catalyst Pharmaceutical Partners, Inc., which has begun Phase I clinical trials [209].

Novel targets are required for the treatment of these complex neurological disorders and progression of GABA modulators to clinical trials suggest fruitful efforts in the right direction. We are quite hopeful that some of them will emerge as novel therapeutics in future.

Index of applied abbreviations and acronyms

AC

American cockroach

AMH

3-acetylamino-4’-O-methylhonokiol

Arg

Arginine

AUC

Area under the curve

bbsP2C

Bovine brain stem cells

BDC

Bile duct-cannulated

BDZ

Benzodiazepines

bfcP2B

Brain frontal cortex cells

BGT

GABA uptake protein

BMI

Body mass index

C188

Cysteine

CBR

Central Benzodiazepines receptors

CC

Common cutworm

CHO

Chinese hamster ovary

Cmax

Maximum concentration

CNS

Central Nervous System

D162

Aspartic Acid

DMCM

Methyl-6,7-dimethoxy-4-ethyl-β- carboline-3-carboxylate

DMSO

Dimethyl sulfoxide

EBOB

4-ethynyl-4-n-propylbicycloorthobenzoate

EC₅₀

50% effective concentration

ED₅₀

Effective dose

FLIPR

Fluorometric imaging plate reader

FMP

Fluorometric membrane potential

Fe

Ferrocene

GABA

Gamma-aminobutyric acid

GABA_A-R

GABA-A receptors

GABA-AT

GABA Amino transferees

GABACls

GABA induced chloride current

GAT

GABA transporters

GB

GABA binding

GHB

Gamma-hydroxybutyric acid

GIRK

G-protein activated inwardly rectifying

Glu

Glutamic acid

GluCls

Glutamate–gated chloride channels

HEK293

Human embryonic kidney cells

HPMC

Hydroxypropylmethylcellulose

i.v.

Intra venous

IC₅₀

50% inhibitory concentration

ID/g

Injected dose per gram

I_GABA-max

Maximal GABA-induced chloride current modulation

log D

Lipophilic parameter

log P

Lipophilic parameter

mGABA

Mammalian GABA

mGAT

Mammalian GABA transferase

MH

4’-O-methylhonokiol

nAChRs

Nicotinic acetylcholine receptors

NAM

Negative Allosteric Modulation

nM

Nano molar

OA

Open arm

PAM

Positive Allosteric Modulation

PET

Positron emission tomography

Phe

Phenylalanine

po

per os (By mouth)

PTZ

Pentilenetetrazoleas

RDL

Resistance to dieldrin

SAR

Structure activity relationship

SBP

Small brown plant

Ser

Serine

SIH

Stress induced hyperthermia

SMD

Steered molecular dynamics

SPV

Saccadic peak velocity

STZ

Streptozotocin

TBPS

tert-butylbicyclophosphorothionates

Thr

Threonine

TPMPA

tetrahydropyridine-4-yl-methyl-phosphinic acid

Tyr

Tyrosine